The long-term goal of the proposed research is to develop a novel multimodal approach for optical staging of epithelial precancerous lesions and early neoplasms using the nonlinear optical imaging techniques of multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM). The research will combine the ability to detect and map endogenous tissue autofluorescence in the epithelium and stroma assessed by MPM with second harmonic generation originating specifically from stromal structural proteins, primarily collagen. Significant optical and microstructural alterations have been documented with neoplastic transformation. However, while a number of previous optical methods have been associated with high sensitivity to neoplastic change, they generally lacked specificity in detection and staging. It is anticipated that the ability to assess both microarchitectural and optical changes in-depth by MPM-SHGM, resulting in image representations on a level similar to histology, will result in higher specificity than previous techniques. The MPM-SHGM technique will be developed and refined in a model of oral squamous cell carcinoma (which occurs in a site suited for the development of optical assessment techniques) and will be applicable to other epithelial neoplasms at various tissue sites in the future. In brief, the project will involve the a) the spatiotemporal study of epithelial neoplastic transformation in vivo in order to better understand alterations in endogenous optical signals revealed by MPM-SHGM and to identify significant image-based parameters which may serve as markers of neoplastic progression, b) the development and refinement of a noninvasive staging methodology by MPM-SHGM which may assist in guiding surgical resection and in surveillance of patients at risk for recurrence, and c) evaluation of the combination of exogenous contrast agents with MPM-SHGM to reveal molecular-specific signal alterations indicative of neoplastic potential. This research addresses a significant problem in staging of epithelial neoplasms. There is a need for highly specific noninvasive detection methods to assist in the early staging of epithelial neoplasms in general because the chances of patient survival are directly correlated to the stage at which epithelial cancers are detected and treated. Oral cancer, the 6th most common cancer in the world and one which is associated with a five-year survival rate of only 50-55%, is associated with a significant local recurrence rate for which early detection and staging is of primary importance to patient survival. Despite occurring in a highly accessible tissue for inspection high mortality and morbidity rates, particularly following recurrence, indicate the need for improved detection and staging of oral neoplasia.